Construction method of fingerprint spectrum of Shenkun blood-nourishing granules and fingerprint spectrum of Shenkun blood-nourishing granules

Abstract

The invention discloses a construction method of a fingerprint spectrum of Shenkun blood nourishing granules and the fingerprint spectrum of the Shenkun blood nourishing granules, and belongs to the technical field of traditional Chinese medicine detection. Different batches of Shenkun blood nourishing particles are adopted to prepare a test solution; the preparation method comprises the following steps: preparing a single reference substance solution from reference substances of tanshinol, leonurine hydrochloride, syringin, protocatechualdehyde, caffeic acid, calycosin-7-O-beta-D-glucoside, lobetyolin, formononin, rosmarinic acid, alkannic acid, luteolin and kaempferol; respectively performing high performance liquid chromatography and high resolution mass spectrometry on the test solution and the reference substance solution, totally screening 18 characteristic peaks, and totally identifying 12 chemical components through standard substance comparison and fragment information analysis to obtain the fingerprint spectrum of the Shenkun blood nourishing granules. The fingerprint spectrum of the Shenkun blood-nourishing granules established by the method can be used for quality control detection of the Shenkun blood-nourishing granules, is beneficial to comprehensive monitoring of product quality, and ensures consistency, controllability and stability of the product quality.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a method for constructing a fingerprint spectrum of Shenkun Yangxue granules and a fingerprint spectrum thereof. Background Art

[0002] Shenkun Yangxue Granules are composed of six medicinal herbs: astragalus, salvia miltiorrhiza, codonopsis pilosula, angelica, motherwort and Patrinia scabra. They have the effects of nourishing qi and blood, promoting blood circulation and removing blood stasis. They are often used clinically to treat symptoms such as postpartum lochia and lower abdominal pain caused by qi deficiency and blood stasis.

[0003] In recent years, Shenkun Yangxue Granules, as a traditional Chinese medicine preparation, has attracted widespread attention due to its significant efficacy and low side effects. However, due to the complex composition of traditional Chinese medicine and the diverse interaction mechanisms between the components, the quality control of Shenkun Yangxue Granules has posed a huge challenge. At present, the quality research of Shenkun Yangxue Granules is limited to the analysis of the single medicinal ingredients in the formula, while ignoring the comprehensive consideration of the overall composition. The current quality standards only include the identification of the prescription Chinese medicinal materials by thin layer chromatography and the determination of protocatechuic aldehyde by high performance liquid chromatography. However, the chemical composition of Shenkun Yangxue Granules is complex. It is difficult to fully reflect its material basis and chemical composition information by relying only on the identification or content determination of a single compound, and thus it is impossible to achieve comprehensive control of its intrinsic quality, which may affect the stability and safety of its clinical effect. Therefore, in order to improve the quality control level of Shenkun Yangxue Granules, it is urgent to develop more comprehensive and systematic detection technologies to achieve a comprehensive evaluation of the overall composition of the granules. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a method for constructing a fingerprint spectrum of Shenkun Yangxue Granules and its fingerprint spectrum, so as to solve the technical problem that the existing Shenkun Yangxue Granule detection method cannot perform comprehensive quality control.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention discloses a method for constructing a fingerprint spectrum of Shenkun Yangxue Granules, comprising the following steps:

[0007] S1. Prepare the test solution: weigh different batches of Shenkun Yangxue Granule powder respectively, add extraction solvent, reflux extraction, and obtain the test solution;

[0008] S2. Prepare reference solution: accurately weigh danshensu, leonurine hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, calycosin 7-O-β-D-glucoside, codonopsis pilosula, formononetin, rosmarinic acid, lithospermic acid, luteolin and kaempferol, and dissolve them to obtain a single reference solution;

[0009] S3, performing chromatographic analysis on the test solution obtained in S1 and the reference solution obtained in S2, respectively, and recording the corresponding chromatograms;

[0010] S4, importing the chromatogram obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system for similarity analysis;

[0011] S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain a total ion flow diagram and a mass spectrum result diagram of the chemical components. Import the test data into Xcalibur software, enter the Qual Browser interface, perform data analysis based on the peak conditions of the chemical components, determine the chemical components of each peak in the test sample chromatogram based on the total ion flow diagram and the mass spectrum result diagram of the chemical components in combination with the reference sample chromatogram, and obtain the fingerprint spectrum of Shenkun Yangxue Granules.

[0012] Preferably, in S1, the extraction solvent is pure methanol solution.

[0013] Preferably, in S1, reflux extraction is performed for 30 min.

[0014] Preferably, in S1, the dosage ratio of the fine powder of Shenkun Yangxue Granules to the extraction solvent is: 3.5g:25mL.

[0015] Preferably, in S2, the preparation method of the reference solution is: accurately weigh danshensu, leonurine hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, calycosin 7-O-β-D-glucoside, codonopsis glycoside, formononetin, rosmarinic acid, lithospermic acid, luteolin and kaempferol reference substances respectively, add pure methanol solution to prepare a single reference solution containing 56 μg danshensu, 27 μg leonurine hydrochloride, 19 μg syringin, 37 μg protocatechuic aldehyde, 26 μg caffeic acid, 11 μg calycosin 7-O-β-D-glucoside, 16 μg codonopsis glycoside, 89 μg formononetin, 25 μg rosmarinic acid, 49 μg lithospermic acid, 6 μg luteolin or 19 μg kaempferol per 1 mL.

[0016] Preferably, in S3, the liquid chromatography conditions are: chromatographic column model Hedera ODS-2-C18, 4.6×250nm×5μm; DAD detector, detection wavelength 210nm; column temperature 30°C; injection volume: 10μL;

[0017] Further preferably, in the liquid chromatography conditions: the mobile phase is acetonitrile-0.01% phosphoric acid aqueous solution, the flow rate is 1.0 mL / min, and the gradient elution program is: 0-5 min, acetonitrile volume 2%; 5-10 min, acetonitrile volume 2%-13%; 10-20 min, acetonitrile volume 13%-22%; 20-25 min, acetonitrile volume 22%-32%; 25-33 min, acetonitrile volume 32%-49%; 33-39 min, acetonitrile volume 49%-46%; 39-40 min, acetonitrile volume 46%-60%.

[0018] Preferably, in S5, the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, capillary temperature 300°C, auxiliary gas temperature 300°C, scanning mode is full scan mode, and the mass-to-charge ratio scanning range m / z is 100-1500.

[0019] Preferably, in S5, the chemical components of each peak in the chromatogram of the test sample are determined according to the total ion current graph and the mass spectrometry result graph of the chemical components in combination with the chromatogram of the reference substance, which are: Peak 3 is danshensu, Peak 4 is leonurine hydrochloride, Peak 5 is syringin, Peak 6 is protocatechuic aldehyde, Peak 7 is caffeic acid, Peak 9 is calycosin 7-O-β-D-glucoside, Peak 11 is codonopsis pilosula glycoside, Peak 13 is formononetin, Peak 14 is rosmarinic acid, Peak 16 is lithospermic acid, Peak 17 is luteolin, and Peak 18 is kaempferol, thereby obtaining the fingerprint of Shenkun Yangxue Granules.

[0020] The second aspect of the present invention discloses the fingerprint spectrum of Shenkun Yangxue Granules obtained by the above-mentioned construction method.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The invention provides a method for constructing a fingerprint spectrum of Shenkun Yangxue Granules. 1) when preparing a test solution, a reflux extraction method is selected, the extraction effect is good, the peak information is more, and the chromatogram components are relatively comprehensive; 2) when preparing a reference solution, danshensu, leonurine hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, calycosin 7-O-β-D-glucoside, codonopsis pilosula, formononetin, rosmarinic acid, lithospermic acid, luteolin and kaempferol are used as reference substances, which can provide a benchmark for the analysis and comparison of the fingerprint spectrum of Shenkun Yangxue Granules, and ensure the accuracy and reliability of the constructed spectrum; 3) through similarity analysis, the consistency and stability of the quality of traditional Chinese medicine are ensured; 4) the chromatographic analysis is combined with the high-resolution mass spectrometry analysis to ensure the comprehensiveness of the method. The HPLC fingerprint of Shenkun Yangxue Granules was established by simultaneously measuring 15 batches of Shenkun Yangxue Granules to obtain the spectrum, and 12 chemical components were identified by high-resolution mass spectrometry. The obtained chromatograms have high similarity, which can effectively and comprehensively detect Shenkun Yangxue Granules. The stability, repeatability and precision were calculated by its relative retention time and relative peak area. The results showed that the RSD values ​​of the relative retention time and relative peak area were all less than 3%, indicating that this method has the characteristics of good stability, high precision and good repeatability, and can accurately, clearly, comprehensively and objectively evaluate the quality of Shenkun Yangxue Granules. It has significant importance and practical value for effectively controlling the quality of Shenkun Yangxue Granules and ensuring clinical efficacy, and provides quality assurance for clinical efficacy. By establishing the fingerprint of Shenkun Yangxue Granules to ensure the stable performance of its efficacy, it can further promote the safety and effectiveness of Shenkun Yangxue Granules and provide strong guarantees for the clinical application and promotion of Shenkun Yangxue Granules.

[0023] Furthermore, when preparing the test solution, pure methanol solution was used as the extraction solvent, and the extract chromatogram had the most information, the highest component content, and the best extraction effect.

[0024] Furthermore, reflux extraction for 30 minutes can shorten the extraction time and reduce the extraction cost while ensuring the extraction effect.

[0025] Furthermore, when performing chromatographic analysis, the wavelength was selected to be 210 nm, the chromatogram contained the most comprehensive information and the baseline was stable. The column temperature was selected to be 30°C, and the separation effect of each component was better.

[0026] Furthermore, when chromatographic analysis is performed, octadecylsilane bonded silica gel is used as a filler and acetonitrile-0.01% phosphoric acid aqueous solution is used as a mobile phase. A large number of peaks are produced, and the peak shape and separation effect are good. When the flow rate is selected as 1.0 ml / min, the separation of each peak in the fingerprint spectrum is good. When the gradient elution program is selected, the separation is good, the peak shape is better, the baseline is stable, and the chromatographic information is complete. By adopting specific elution conditions, while shortening the detection time, the separation effect of multiple active ingredients is significantly improved, so that more characteristic peaks are included in the fingerprint spectrum, which greatly enriches the spectrum information. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0028] Figure 1 This is the chromatogram obtained in Example 1 during the preparation of the test solution of Shenkun Yangxue Granules of the present invention;

[0029] Figure 2 The chromatogram (A) and mass spectrum (B) of Danshensu reference substance of the present invention are shown;

[0030] Figure 3 The chromatogram (A) and mass spectrum (B) of the leonurine hydrochloride reference substance of the present invention are shown;

[0031] Figure 4 The chromatogram (A) and mass spectrum (B) of syringin of the present invention are shown;

[0032] Figure 5 The chromatogram (A) and mass spectrum (B) of protocatechuic aldehyde of the protocatechuic aldehyde reference substance of the present invention are shown in FIG.

[0033] Figure 6 The caffeic acid chromatogram (A) and caffeic acid mass spectrum (B) of the present invention;

[0034] Figure 7 The chromatogram of the reference substance of calycosin 7-O-β-D-glucoside of the present invention (A) and the mass spectrum of calycosin 7-O-β-D-glucoside (B);

[0035] Figure 8 The chromatogram (A) and mass spectrum (B) of the codonopsis ginsenoside reference substance of the present invention are shown;

[0036] Fig. 9 The chromatogram (A) and mass spectrum (B) of formononetin reference substance of the present invention are shown;

[0037] Fig.10The chromatogram (A) and mass spectrum (B) of rosmarinic acid reference substance of the present invention are shown;

[0038] Fig.11 The chromatogram (A) and mass spectrum (B) of lithospermic acid reference substance of the present invention are shown;

[0039] Fig.12 The chromatogram of the luteolin reference substance of the present invention (A) and the mass spectrum of luteolin (B);

[0040] Fig.13 The chromatogram (A) and mass spectrum (B) of benzoic acid of the kaempferol reference substance of the present invention are shown;

[0041] Fig.14 This is the total ion current diagram of negative ions of the mass spectrum of the Cankun Yangxue Granule of the present invention;

[0042] Fig.15 This is the total positive ion current diagram of the mass spectrum of the Cankun Yangxue Granule of the present invention;

[0043] Fig.16 This is the fingerprint spectrum of the test sample of 15 batches of Shenkun Yangxue Granules of the present invention;

[0044] Fig.17 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test solution of the present invention;

[0045] Fig.18 This is a chromatogram obtained by optimizing the extraction time during the preparation of the test solution of the present invention;

[0046] Fig.19 The chromatogram obtained by optimizing the mobile phase composition under the chromatographic conditions of the present invention;

[0047] Fig. 20 The chromatogram obtained by optimizing the detection wavelength under the chromatographic conditions of the present invention;

[0048] Fig.21 The chromatogram obtained by optimizing the flow rate under the chromatographic conditions of the present invention;

[0049] Fig. 22 The chromatogram obtained by optimizing the column temperature in the chromatographic conditions of the present invention;

[0050] Fig.23 The chromatogram obtained by optimizing the elution program under the chromatographic conditions of the present invention. DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0052] The present invention provides a method for constructing a fingerprint spectrum of Shenkun Yangxue granules, comprising the following steps:

[0053] S1. Prepare the test solution: weigh different batches of Shenkun Yangxue Granule fine powder respectively, add the extraction solvent solution, and reflux extraction to obtain the Shenkun Yangxue Granule test solution;

[0054] Wherein, the extraction solvent solution is a pure methanol solution, and the dosage ratio of the fine powder of Shenkun Yangxue Granules to the pure methanol solution is: 3.5g:25mL;

[0055] S2. Prepare reference substance solution: accurately weigh each reference substance, add pure methanol solution, and prepare a single reference substance solution containing 56 μg danshensu, 27 μg leonurine hydrochloride, 19 μg syringin, 37 μg protocatechuic aldehyde, 26 μg caffeic acid, 11 μg calycosin 7-O-β-D-glucoside, 16 μg codonopsis glycoside, 89 μg formononetin, 25 μg rosmarinic acid, 49 μg lithospermic acid, 6 μg luteolin, and 19 μg kaempferol per 1 mL;

[0056] S3, inject the test solution obtained in S1 and the reference solution in S2 into the high performance liquid chromatograph for chromatographic analysis. The liquid chromatography conditions are: chromatographic column model Hedera ODS-2-C18 (4.6×250nm×5μm); using DAD detector, detection wavelength is 210nm; column temperature is 30℃; flow rate: 1.0mL / min; injection volume: 10μL; mobile phase is acetonitrile (A)-0.01% phosphoric acid aqueous solution (B), and the gradient elution program is: 0-5min, 2%A; 5-10min, 2%-13%A; 10-20min, 13%-22%A; 20-25min, 22%-32%A; 25-33min, 32%-49%A; 33-39min, 49%-46%A; 39-40min, 46%-60%A, good separation of each chromatographic peak in the fingerprint can be achieved, and the corresponding chromatogram is recorded;

[0057] S4, importing the chromatogram obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system, selecting the chromatographic peaks existing in the chromatograms of different batches of Shenkun Yangxue Granules as common peaks, and performing similarity analysis on the chromatograms of the test solution after data import, multi-point correction and data matching respectively;

[0058] S5. The test solution was subjected to high-resolution mass spectrometry analysis. The high-resolution mass spectrometry detection conditions were: electrospray ionization, spray voltage 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, capillary temperature 300°C, auxiliary gas temperature 300°C, full scan mode, mass-to-charge ratio scanning range m / z 100-1500, and the total ion flow diagram and mass spectrometry result diagram of the chemical components were obtained. The test data was imported into Xcalibur software and entered into the Qual Browser interface. Data analysis was performed according to the peak conditions of chemical components. The chemical components of each peak in the test sample chromatogram were determined according to the total ion flow diagram and the mass spectrum results of the chemical components combined with the chromatogram of the reference substance. The results were as follows: Peak 3 was danshensu, Peak 4 was leonurine hydrochloride, Peak 5 was syringin, Peak 6 was protocatechuic aldehyde, Peak 7 was caffeic acid, Peak 9 was calycosin 7-O-β-D-glucoside, Peak 11 was codonopsis pilosula glycoside, Peak 13 was formononetin, Peak 14 was rosmarinic acid, Peak 16 was lithospermic acid, Peak 17 was luteolin, and Peak 18 was kaempferol. The fingerprint of Shenkun Yangxue Granules was obtained.

[0059] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0060] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, are conventional commercial products, and their specifications are conventional specifications in the art.

[0061] 1. Instrument

[0062] The instruments used in the present invention are shown in Table 1.

[0063] Table 1 Instruments used in the present invention

[0064]

[0065] 2. Drugs and reagents

[0066] The 15 batches of Shenkun Yangxue Granule samples used in the present invention were all provided by Shaanxi Bailu Pharmaceutical Co., Ltd. (Table 2). The prescription originated from the surgical gynecology volume of the National Compilation of Chinese Patent Medicine Standards of the State Food and Drug Administration [WS-11309 (ZD-1309) -2002-2012Z]; reference substances: rosmarinic acid reference substance (batch number: C14900895, purity: 97%) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; codonopsis ginsenoside reference substance (batch number: AF20051902, purity: 98%), leonurine hydrochloride reference substance (batch number: AFBJ2706, purity: 98%) were purchased from Chengdu Aifa Biotechnology Co., Ltd.; luteolin reference substance (batch number: 102759, purity: 98%), danshensu reference substance (batch number: YJ0157, purity: 98%) were purchased from Chengdu Aifa Biotechnology Co., Ltd. %) were purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd.; protocatechuic aldehyde reference substance (batch number: 20082402, purity: 99.75%) and calycosin 7-O-β-D-glucoside reference substance (batch number: 21022604, purity: 98%) were purchased from Chengdu Pufeide Biotechnology Co., Ltd.; caffeic acid reference substance (batch number: PS010522, purity: 98%), syringin reference substance (batch number: PS010261, purity: 98%), formononetin reference substance (batch number: PS000671, purity: 98%), lithospermic acid reference substance (batch number: PS001156, purity: 98%) and kaempferol reference substance (batch number: PS011676, purity: 98%) were purchased from Chengdu Pusi Biotechnology Co., Ltd.; the reagents used in the present invention are shown in Table 3.

[0067] Table 2 Samples used

[0068]

[0069] Table 3 Reagents used

[0070]

[0071] The present invention is described in detail below by taking Shenkun Yangxue Granules as an example through specific embodiments.

[0072] Example 1

[0073] A method for constructing a fingerprint spectrum of Shenkun Yangxue granules comprises the following steps:

[0074] S1. Preparation of test solution: accurately weigh 3.5 g of 15 batches of Shenkun Yangxue Granules, place them in stoppered conical flasks respectively, add 25 mL of pure methanol solution, stopper them tightly, reflux and extract for 30 min, shake well, filter, and pass the filtrate through a 0.45 μm microporous filter membrane to obtain the Shenkun Yangxue Granule test solution.

[0075] S2. Preparation of reference solution: Accurately weigh each reference substance (danshensu, leonurine hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, calycosin 7-O-β-D-glucoside, codonopsis glycoside, formononetin, rosmarinic acid, lithospermic acid, luteolin and kaempferol), respectively add pure methanol solution to prepare a single reference solution containing 56 μg of danshensu, 27 μg of leonurine hydrochloride, 19 μg of syringin, 37 μg of protocatechuic aldehyde, 26 μg of caffeic acid, 11 μg of calycosin 7-O-β-D-glucoside, 16 μg of codonopsis glycoside, 89 μg of formononetin, 25 μg of rosmarinic acid, 49 μg of lithospermic acid, 6 μg of luteolin and 19 μg of kaempferol per 1 mL.

[0076] S3, accurately pipette 10 μL of the test solution obtained in S1 and the reference solution in S2, respectively, inject them into the high performance liquid chromatograph, perform chromatographic analysis, and record the corresponding chromatograms, such as Figure 1 as well as Figure 2 to Figure 13 As shown in (A).

[0077] The liquid chromatography conditions are as follows: chromatographic column: Hedera ODS-2-C18 (4.6×250nm×5μm) chromatographic column; detector: DAD detector; detection wavelength: 210nm; flow rate: 1.0mL / min; injection volume: 10μL; column temperature: 30°C; mobile phase: acetonitrile-0.01% phosphoric acid aqueous solution, gradient elution, the elution program is shown in Table 11:

[0078] S4. The chromatogram obtained in S3 was imported into the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, and the chromatographic peaks existing in the chromatograms of 15 batches of Shenkun Yangxue Granules were selected as common peaks. The chromatograms of the test solution were subjected to data import, multi-point correction and data matching, and similarity analysis was performed (Table 4) to confirm the reliability of the results.

[0079] Table 4 Similarity between each batch of samples of Shenkun Yangxue Granules and the common pattern

[0080]

[0081] S5. To determine the chemical components in the fingerprint, the above sample solution was subjected to mass spectrometry analysis. The high-resolution mass spectrometry detection conditions were: electrospray ionization, spray voltage 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, mass-to-charge ratio scanning range m / z 100-1500, and the total ion flow diagram and mass spectrum result diagram of the chemical components were obtained ( Figure 2 to Figure 13 Middle picture (B), Fig.14 as well as Fig.15 ).

[0082] S6. Import the test data into Xcalibur software, enter the Qual Browser interface, perform data analysis according to the chemical component peaks, and determine the chemical components of each peak in the test sample chromatogram according to the total ion flow diagram and the mass spectrum result diagram of the chemical components combined with the reference sample chromatogram. Among them, peak 3 is danshensu, with a retention time of 14.357 min; peak 4 is leonurine hydrochloride, with a retention time of 15.167 min; peak 5 is syringin, with a retention time of 16.793 min; peak 6 is protocatechuic aldehyde, with a retention time of 17.950 min; peak 7 is caffeic acid, with a retention time of 20.360 min; 9 Peak 1 is calycosin 7-O-β-D-glucoside, with a retention time of 24.140 min; Peak 11 is codonopsis ginsenoside, with a retention time of 28.137 min; Peak 13 is formononetin, with a retention time of 29.347 min; Peak 14 is rosmarinic acid, with a retention time of 29.677 min; Peak 16 is lithospermic acid, with a retention time of 31.823 min; Peak 17 is luteolin, with a retention time of 32.227 min; Peak 28 is kaempferol, with a retention time of 35.370 min, and the fingerprint of Shenkun Yangxue Granules was obtained ( Fig.16 ).

[0083] S7. Methodological considerations

[0084] 1. Precision experiment

[0085] Take the test solution obtained in S1 and inject it in parallel 6 times according to the chromatographic conditions in S3, with an injection volume of 10 μL. Take danshensu, leonurine hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, calycosin 7-O-β-D-glucoside, codonopsis pilosula, formononetin, rosmarinic acid, lithospermic acid, luteolin and kaempferol as reference peaks. By analyzing the relative retention time and relative peak area, the RSD value was calculated. The results showed that the RSDs were 0.18%-0.33% and 0.84%-2.42%, respectively, and the RSDs were all less than 3%, indicating that the precision of the instrument was good.

[0086] 2. Stability test

[0087] The test solution obtained in S1 was taken, and according to the chromatographic conditions in S3, samples were injected and analyzed at 0h, 2h, 4h, 8h, 12h and 24h, respectively, with an injection volume of 10μL. Danshensu, leonurine hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, calycosin 7-O-β-D-glucoside, codonopsis pilosula, formononetin, rosmarinic acid, lithospermic acid, luteolin and kaempferol were used as reference peaks. The relative retention time and relative peak area of ​​the common peaks in the HPLC fingerprint of the samples were analyzed and the RSD value was calculated. The results showed that the RSDs were 0.35% to 0.69% and 0.93% to 2.31%, respectively, and the RSDs were all less than 3%, indicating that the test solution of Shenkun Yangxue Granules had good stability within 24h.

[0088] 3. Repeatability Experiment

[0089] Six portions of Shenkun Yangxue Granule test solution were taken and analyzed separately according to the chromatographic conditions in S3. Danshensu, leonurine hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, calycosin 7-O-β-D-glucoside, codonopsis pilosula, formononetin, rosmarinic acid, lithospermic acid, luteolin and kaempferol were used as reference peaks. The relative retention time and relative peak area of ​​the common peaks in the HPLC fingerprint of the samples were analyzed and the RSD values ​​were calculated. The results showed that the RSDs were 0.28%-0.71% and 0.66%-2.79%, respectively, and the RSDs were all less than 3%, indicating that the method had good repeatability.

[0090] Example 2

[0091] Optimization of the preparation of the test solution: The purpose of this example is to investigate the effects of different extraction methods (ultrasound, reflux, immersion) on the detection of the test solution of Shenkun Yangxue Granules. The chromatographic conditions are the same as those in Example 1, and the preparation conditions of the remaining test solutions are the same as those in Example 1. The test results are shown in FIG. Fig.17 As shown. Fig.17 It can be seen that when the extraction method is reflux, the extraction effect is better, the peak information is more, and the chromatogram components are more comprehensive.

[0092] Example 3

[0093] Optimization of the preparation of the test solution: The purpose of this example is to investigate the effect of different extraction times (30min, 60min, 90min) on the detection of the test solution of Shenkun Yangxue Granules. The chromatographic conditions are the same as those in Example 1, and the preparation conditions of the other test solutions are the same as those in Example 1. The test results are as follows: Fig.18 As shown. Fig.18 It can be seen that the test results of extraction time of 30min, 60min and 90min are not much different. Considering that 30min saves more time and cost, 30min is selected as the extraction time condition.

[0094] Example 4

[0095] Optimization in the preparation of the test solution: The purpose of this example is to investigate the effect of different extraction solvents (methanol, ethanol, acetonitrile, water, 0.01% phosphoric acid water) on the detection of the test solution of Shenkun Yangxue Granules. The chromatographic conditions are the same as those in Example 1, and the preparation conditions of the remaining test solutions are the same as those in Example 1. The test results show that methanol extraction has the best effect.

[0096] Example 5

[0097] Optimization in the preparation of the test solution: The purpose of this example is to investigate the effect of different extraction solvent concentrations (50% methanol solution, 80% methanol solution, pure methanol solution) on the detection of the Shenkun Yangxue Granule test solution. The chromatographic conditions are the same as in Example 1, and the preparation conditions of the remaining test solutions are the same as in Example 1. The test results show that when pure methanol solution is used as the extraction solvent, the extract chromatogram has the most information and the highest component content.

[0098] Example 6

[0099] Optimization of chromatographic conditions: The purpose of this example is to investigate the effects of different mobile phases (methanol-0.01% phosphoric acid, acetonitrile-0.1% phosphoric acid, acetonitrile-0.05% phosphoric acid, acetonitrile-0.02% phosphoric acid, acetonitrile-0.01% phosphoric acid, acetonitrile-0.05% acetic acid, acetonitrile-0.1% formic acid) on the detection of the test solution of Shenkun Yangxue Granules. The test solution was prepared by the method of Example 1. The test results are shown in FIG. Figure 2 As shown. Fig.19 It can be seen that when the mobile phase is acetonitrile-0.01% phosphoric acid, the number of peaks is larger, and the peak shape and separation effect are better.

[0100] Example 7

[0101] The purpose of this example is to investigate the effect of different wavelengths (190nm, 210nm, 245nm, 275nm, 280nm, 310nm) on the detection of the test solution of Shenkun Yangxue Granules. The test solution was prepared by the method of Example 1, and the other chromatographic conditions were the same as those of Example 1. The test results are shown in Fig. 20 As shown. Fig. 20 It can be seen that when the wavelength is 210nm, the chromatogram contains the most comprehensive information and the baseline is stable, so this method is selected as the detection wavelength condition.

[0102] Example 8

[0103] The purpose of this example is to investigate the effect of different flow rates (0.6 ml / min, 0.8 ml / min, 1.0 ml / min) on the detection of the test solution. The test solution was prepared by the method of Example 1, and the other chromatographic conditions were the same as those of Example 1. The test results are shown in Fig.21 As shown. Fig.21 It can be seen that the separation of the peaks in the fingerprint spectrum is better when the flow rate is 1.0 ml / min, so the flow rate of 1.0 ml / min is selected as the flow rate condition.

[0104] Example 9

[0105] The purpose of this example is to investigate the effect of different column temperatures (25°C, 30°C, 35°C) on the detection of the test solution of Shenkun Yangxue Granules. The test solution was prepared by the method of Example 1, and the other chromatographic conditions were the same as those of Example 1. The test results are shown in Fig. 22 As shown. Fig. 22 It can be seen that the separation effect of each component is better when the column temperature is 30℃, so the column temperature is selected to be 30℃.

[0106] Example 10

[0107] The purpose of this example is to investigate the effect of different elution procedures on the detection of the test solution of Shenkun Yangxue Granules. The test solution was prepared by the method of Example 1, and the elution procedures were set respectively. The other chromatographic conditions were the same as those in Example 1. Some elution procedures are shown in Tables 5 to 11:

[0108] Table 5 Elution program 1

[0109]

[0110] Table 6 Elution Program 2

[0111]

[0112] Table 7 Elution Program 3

[0113]

[0114] Table 8 Elution Program 4

[0115]

[0116] Table 9 Elution Program 5

[0117]

[0118] Table 10 Elution Program 6

[0119]

[0120] Table 11 Elution Program 7

[0121]

[0122] Test results such as Fig.23 As shown. Fig.23 It can be seen that elution program 7 has good separation, better peak shape, stable baseline, and complete chromatographic information. Therefore, elution program 7 is selected as the optimal elution program.

[0123] The above experimental results show that this method has the characteristics of good stability, high precision and good repeatability, and can comprehensively and objectively evaluate the quality of Shenkun Yangxue Granules and provide quality assurance for clinical efficacy.

[0124] The present invention screens the extraction solvent, extraction method, mobile phase, column temperature, flow rate and other conditions, uses octadecylsilane bonded silica gel as a filler, acetonitrile and phosphoric acid aqueous solution as mobile phases, and adopts specific elution conditions. While shortening the detection time, it also significantly improves the separation effect of multiple effective ingredients, so that more characteristic peaks are included in the fingerprint spectrum, which greatly enriches the spectrum information, and 12 chemical components are jointly identified by high-resolution mass spectrometry, which can effectively and comprehensively detect the Shenkun Yangxue Granules. The present invention establishes a fingerprint spectrum quality evaluation method for Shenkun Yangxue Granules for the first time, and uses this method to comprehensively detect the types and quantities of the main chemical components contained in the Chinese patent medicine Shenkun Yangxue Granules, which can objectively and comprehensively evaluate the quality of Shenkun Yangxue Granules in a fast, convenient and accurate manner.

[0125] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of Shenkun Yangxue Granules, characterized in that: The following steps are involved: S1. Prepare the test solution: weigh different batches of Shenkun Yangxue Granule powder respectively, add extraction solvent, reflux extraction, and obtain the test solution; S2. Prepare reference solution: accurately weigh danshensu, leonurine hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, calycosin 7-O-β-D-glucoside, codonopsis pilosula, formononetin, rosmarinic acid, lithospermic acid, luteolin and kaempferol, and dissolve them to obtain a single reference solution; S3, performing chromatographic analysis on the test solution obtained in S1 and the reference solution obtained in S2, respectively, and recording the corresponding chromatograms; S4, importing the chromatogram obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system for similarity analysis; S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain a total ion flow diagram and a mass spectrum result diagram of the chemical components. Import the test data into the Xcalibur software and enter the QualBrowser interface to perform data analysis based on the peaks of the chemical components. Determine the chemical composition of each peak in the chromatogram of the test sample based on the total ion flow diagram and the mass spectrum result diagram of the chemical components in combination with the chromatogram of the reference sample to obtain the fingerprint of Shenkun Yangxue Granules.

2. The method for constructing a fingerprint spectrum of Shenkun Yangxue Granule according to claim 1, characterized in that: In S1, the extraction solvent is pure methanol solution.

3. The method for constructing a fingerprint spectrum of Shenkun Yangxue Granule according to claim 1, characterized in that: In S1, reflux extraction was performed for 30 min.

4. The method for constructing a fingerprint spectrum of Shenkun Yangxue Granule according to claim 1, characterized in that: In S1, the dosage ratio of the fine powder of Shenkun Yangxue Granules to the extraction solvent is: 3.5g:25mL.

5. The method for constructing a fingerprint spectrum of Shenkun Yangxue Granule according to claim 1, characterized in that: In S2, the preparation method of the reference solution is as follows: accurately weigh danshensu, leonurine hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, calycosin 7-O-β-D-glucoside, codonopsis glycoside, formononetin, rosmarinic acid, lithospermic acid, luteolin and kaempferol reference substances respectively, add pure methanol solution to prepare a single reference solution containing 56 μg of danshensu, 27 μg of leonurine hydrochloride, 19 μg of syringin, 37 μg of protocatechuic aldehyde, 26 μg of caffeic acid, 11 μg of calycosin 7-O-β-D-glucoside, 16 μg of codonopsis glycoside, 89 μg of formononetin, 25 μg of rosmarinic acid, 49 μg of lithospermic acid, 6 μg of luteolin or 19 μg of kaempferol per 1 mL.

6. The method for constructing a fingerprint spectrum of Shenkun Yangxue Granule according to claim 1, characterized in that: In S3, the liquid chromatography conditions are as follows: chromatographic column model Hedera ODS-2-C18, 4.6×250nm×5μm; DAD detector is used, and the detection wavelength is 210nm; column temperature is 30°C; injection volume: 10μL.

7. The method for constructing a fingerprint spectrum of Shenkun Yangxue Granule according to claim 6, characterized in that: Liquid chromatography conditions: mobile phase is acetonitrile-0.01% phosphoric acid aqueous solution, flow rate: 1.0 mL / min, gradient elution program: 0-5 min, acetonitrile volume 2%; 5-10 min, acetonitrile volume 2%-13%; 10-20 min, acetonitrile volume 13%-22%; 20-25 min, acetonitrile volume 22%-32%; 25-33 min, acetonitrile volume 32%-49%; 33-39 min, acetonitrile volume 49%-46%; 39-40 min, acetonitrile volume 46%-60%.

8. The method for constructing a fingerprint spectrum of Shenkun Yangxue Granule according to claim 1, characterized in that: In S5, the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300°C, auxiliary gas temperature 300°C, scanning mode is full scan mode, and the mass-to-charge ratio scanning range m / z is 100-1500.

9. The method for constructing a fingerprint spectrum of Shenkun Yangxue Granule according to claim 1, characterized in that: In S5, the chemical components of each peak in the chromatogram of the test sample were determined according to the total ion flow chart and the mass spectrometry results of the chemical components, combined with the chromatogram of the reference sample, which were: Peak 3 was danshensu, Peak 4 was leonurine hydrochloride, Peak 5 was syringin, Peak 6 was protocatechuic aldehyde, Peak 7 was caffeic acid, Peak 9 was calycosin 7-O-β-D-glucoside, Peak 11 was codonopsis pilosula glycoside, Peak 13 was formononetin, Peak 14 was rosmarinic acid, Peak 16 was lithospermic acid, Peak 17 was luteolin, and Peak 18 was kaempferol, and the fingerprint of Shenkun Yangxue Granules was obtained.

10. The fingerprint of Shenkun Yangxue Granules obtained by the construction method described in any one of claims 1 to 9.

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